CD31 (PECAM-1)
Immune responses can be controlled by inhibitory immune receptors among which CD31 (PECAM-1), which is expressed exclusively and constitutively on cells at the blood-vessel interface.
CD31 consists of a single chain molecule comprising 6 Ig-like extracellular domains, a short transmembrane segment and a cytoplasmic tail containing two ImmunoTyrosine-based Inhibitory Motif (ITIM)s. The structure of CD31 is shown in the table below.
DomainPosition on SEQ ID No: 1Signal peptide 1 to 27Extracellular domain 28 to 601First Ig-like extracellular domain 34 to 121Second Ig-like extracellular domain145 to 233Third Ig-like extracellular domain236 to 315Fourth Ig-like extracellular domain328 to 401Fifth Ig-like extracellular domain424 to 493Sixth Ig-like extracellular domain499 to 591Juxta-membrane domain592 to 601Transmembrane domain602 to 620Cytoplasmic domain621 to 738
The consequent putative immunoregulatory properties of CD31 are supported by the fact that CD31 signaling drives mutual repulsion of blood leukocytes and modulates the balance between inhibitory and stimulatory signals of both innate and adaptive immune cells. Mechanical engagement of the distal Ig-like extracellular domains of CD31 induces outside-in inhibitory signaling triggered by the phosphorylation of its ITIMs, and the recruitment and activation of SH2-containing phosphatases.
Zehnder et al. (1995, Blood. 85(5):1282-8) identified a CD31 antibody that inhibited the mixed lymphocyte reaction (MLR) in a specific and dose-dependent manner. They further found that a CD31 peptide corresponding to the epitope of this antibody, i.e. to the 23 membrane-proximal amino acids of CD31, strongly inhibited the MLR. They hypothesized that the 23 membrane-proximal amino acids of CD31 is a functionally important region, and that the CD31 peptide interferes with lymphocyte activation by competing for binding epitopes. However, Zehnder et al. failed to teach whether CD31-mediated signaling is activated or inhibited by the CD31 peptide.
Chen et al. (1997, Blood. 89(4):1452-9) showed that this peptide delayed onset of graft-versus-host disease (GVHD) and increased long-term survival in a murine model of the disease. They hypothesized that the CD31 peptide inhibits a common pathway in T-cell activation. Again, Chen et al. failed to elucidate the role played by the CD31 peptide in T-cell activation. In particular, these previous works did not assess the putative effect of the peptide on the CD31 signaling cascade and more precisely on the phosphorylation state of the CD31 ITIMs.
For a yet unknown mechanism, CD31 is “lost” on certain circulating lymphocytes. Its loss is observed upon lymphocyte activation and it has been recently shown that the absence of lymphocyte CD31 signaling, in turn, heightens the pathologic immune responses involved in the development of atherothrombosis.
A soluble form of CD31, due to a variant transcript lacking the transmembrane segment, has also been reported and therefore it is currently thought that the individual amount of circulating CD31 is genetically determined. Consequently, a number of previous studies have attempted to find a correlation between plasma levels of soluble CD31 and the risk of atherothrombosis or other autoimmune diseases. However, independently of the specific genetic polymorphisms analyzed, data showed a broad range of plasma CD31 values and the results of these different studies were contradicting.
There is therefore a need for better understanding the biological function of CD31. This would allow the provision of better tools for the diagnosis of diseases linked with T-cell activation such as thrombotic and autoimmune disorders.
Methods for Analyzing Signaling Pathways
Three different assays are currently available for analyzing signaling pathways: co-immunoprecipitation followed by Western Blot (co-IP/WB), the Cellular Activation of Signaling ELISA assay (CASE) and the CBA Flex set (BD) assay.
In the co-IP/WB assay, cells are lysed and a protein extraction is first carried out. The studied protein is then co-immunoprecipitated with proteins with which it is associated and a 2D electrophoresis is carried out. Finally, the membrane obtained after the Western Blot is hybridized with a labelled antibody and the signal is detected. It is thus a time-consuming assay. The membrane may be deshybridized and rehybridized with another labelled antibody, but no more than four times. Thus only four parameters may be analyzed with a single sample/membrane. In addition, the co-IP/WB assay is a qualitative but not a quantitative assay. Finally, the skilled in the art must perform two separate co-IP/WB assays if he wishes to compare proteins associated with the studied protein in a phosphorylated state with those associated with the studied protein in an unphosphorylated state.
The CASE (Superarray) and Phosphlow (BD) assays are simpler and more rapid than the co-IP/WB assay since the labelled antibody is added to a sample comprising permeabilized but not lysed cells. In addition, no blotting is needed. The antibodies are labelled with a fluorophore (Phosphlow) or detected via an enzyme (CASE), the activity of which can easily be measured. However, these assays do still not allow analyzing many parameters with a single sample since only a limited number of enzymes are available. In addition, both assays are much less specific than the co-IP/WB assay since the studied molecule is not captured and therefore one cannot determine the interaction between the different molecules of the signaling pathway. The Phosphlow and CASE assays only allow determining which molecules of a given signaling pathway are present in a cell, or present in a phosphorylated state in the cell.
The CBA Flex set (BD) assay allows simultaneous determination of multiple signaling molecules but fails to allow the analysis of molecular complexes, and therefore, the determination of specific receptor-dependent signaling cascades is impossible.
There is therefore a need for improved methods for analyzing signaling pathways, which combines the respective advantages of co-IP/WB, Phosphlow/CASE and CBA Flex set.